Duchenne Muscular Dystrophy (DMD) is due to the mutation of a gene in the X chromosome coding for a protein called dystrophin (Koenig et al 1987; Hoffman et al. 1987; Bodrug et al. 1987, Arahata et al. 1988, Sugita et al. al 1988). The mutations of the dystrophin vary from one family of patients to another but always lead to the absence of a functional dystrophin protein under the membrane on the muscle fiber (Hoffman et al. 1987; Chelly et al. 1990; Chamberlain et al. 1991; Anderson et al 1992; Kilimann et al. 1992; Roberts et al 1992). The absence of dystrophin leads to an increase vulnerability of the muscle fibers during contraction (Menke 1995). Repeated cycles of damages and repairs produce a progressive reduction of the number of muscle fibers and to loss of strength which confine the patients to a wheel chair by the age of ten and to premature death in their early twenties.
Roughly 70% of the mutations of the dystrophin gene are large deletion of one of several exons (Anderson et al 1992; Kilimann et al. 1992). The other mutations are small point mutations due either to a small deletion of a few base pairs leading to a shift of the reading frame or changes of only one base pair producing a missense or a stop codon (Bullman et al 1991. Corrado et al. 1994; Roberts et al. 1992; Clemens et al 1992; Nicholson et al. 1993). Around 5% of all DMD mutations may be due to stop codons.
Cystic fibrosis (CF) is due to a mutation of a gene coding for the CF transmembrane conductance regulator (CFTR) protein. Howard et al. (1996) made experiments with a bronchial epithelial cell line obtained from a CF patient having a premature stop mutation in the CFTR gene. This mutation resulted in a premature end of the synthesis of the CFTR protein and thus in a non-functional protein. They incubated this cell line with aminoglycoside antibiotics G418 (100 mg/mL) or with a gentamicin (200 mg/mL) during 18 to 24 hours. This incubation with gentamicin permitted to suppress the premature stop mutation by inserting an amino acid at the stop codon. A full-length CFTR protein was thus obtained The suppression of the premature stop codon by gentamicin is mediated by mis-pairing between the stop codon and a near-cognate aminoacyl tRNA. Bedwell et al. (1997) recently demonstrated that this full length CFTR protein resulting from the incubation with the aminoglycoside antibiotics was present in the cell membrane and functional.
The mdx mouse is an animal model for DMD. It has a point mutation in the dystrophin gene resulting in a truncated protein which is not incorporated in the muscle fiber membrane (Hoffman et al 1987).
Therefore, this model is proper for testing the effect of aminoglycosides.
In accordance with the present invention is provided the first method for the in vivo treatment of a disease which is clue to the presence of a premature stop codon in a nucleic acid encoding a protein involved in the etiology of the disease, the method comprising the step of:
administering to the subject an effective dose of an aminoglycoside antibiotic, a derivative thereof or an aminoglycoside-like molecule to suppress the expression of said stop codon.
In a particular embodiment, each mention of the term xe2x80x9caminoglycosidexe2x80x9d is intended to mean xe2x80x9cgentamicinxe2x80x9d. Other related molecules or derivatives are within the scope of this invention.
In a preferred embodiment, the aminoglycoside antibiotic is gentamicin sulfate.
The disease that has been treated in practice is Duchenne Muscular Dystrophy. Other diseases caused by a stop mutation would benefit from this invention.
The effective dose of aminoglycoside can be administered intra-muscularly, intravenously or subcutaneously, preferably intramuscularly.
The effective dose is equivalent to about 8 to 40 mg gentamicin sulfate per kg of body weight per day in mice administered intra-muscularly, or to about 1.5 to 6 mg gentamicin sulfate per kg of body weight per day in humans administered intra-muscularly.
Is therefore contemplated a new use of an aminoglycoside antibiotic, a derivative thereof or an aminoglycoside-like molecule in the making of a medication to treat a subject affected by a disease due to the presence of a premature stop codon in a nucleic acid encoding a protein involved in the etiology of the disease, whereby the expression of said stop codon is suppressed and said nucleic acid is correctly translated in to a functional protein.